Fertilization in mammals occurs through a series of highly ordered steps that lead ultimately to the fusion of a single sperm cell with an ovulated egg. The first step in this process is the binding of free-swimming sperm to the extracellular egg coat, or zona pellucida, through a mechanism which, in general, is highly species-specific. The specificity of this interaction implicates the existence of specific complementary molecules on egg and sperm, referred to respectively as the sperm receptor and the egg binding protein. The primary sperm receptor on mouse eggs has been identified as a single zona glycoprotein - ZP3. In contrast, a number of molecules and enzymes have been proposed as candidates for the sperm surface egg-binding protein. One such candidate has been identified in two assays - by affinity chromatography and by crosslinking to ZP3 - as a Mr 56,000 polypeptide observed on SDS gels. Cloning and characterization has been accomplished for the mouse and human homologs of a gene (called Tcte1) that appears to encode this polypeptide based on functional assays as well as evolutionary considerations. Funds are requested to carry out a series of genetic and evolutionary studies aimed at understanding the function of this gene product in the process of fertilization. Two major goals are to answer the questions: (1) Is the TCTE1 polypeptide necessary for fertilization?, and (2) Is the TCTE1 polypeptide sufficient for the initiation of species-specific fertilization. Targeted mutagenesis will be used to produce mice that do not express the Tcte-1 gene, and transgenic mice will be generated that express TCTE1 from a heterologous species such as hamster, rat, or guinea pig. Studies of fertility and egg binding with these animals will provide definitive answers to the questions that have been posed. A third goal is to determine whether the Tcte1 gene is under positive Darwinian selection, which would be predicted if TCTE1 played a role in establishing post-mating reproductive barriers between species. This question will be approached through comparative sequence analysis, which could also provide insight into the nature of the interaction between TCTE1 and the egg surface. The demonstration of positive Darwinian selection would provide confirming evidence for a species-specific role of TCTE1 in gamete recognition.